Project Summary. Lymphangioleiomyomatosis (LAM) is a rare lung disease of women in which abnormally proliferating smooth muscle cells carrying acquired mutations in the TSC1 or TSC2 genes metastasize from an unknown source and infiltrate the lung. The proliferating smooth muscle cells lead to destruction of surrounding lung tissue, cystic remodeling, and organ failure. Unfortunately, physicians typically diagnose LAM after significant disease progression. We reported key findings regarding immune function [specifically, natural killer (NK) cells] at this stage of disease in LAM patients, but there is still an unmet need to further define the role of these cells in advanced disease as well as early stages of disease. Early events can be effectively studied using animal models that best reflect the pathological processes leading to LAM. Our preliminary data examining LAM patients and a mouse model of LAM featuring leiomyoma formation and Tsc2-null nodules in the lungs demonstrate the existence of organ-specific alterations in NK cell function that likely contribute to LAM pathogenesis. Specifically, unique subsets of NK cells exist in circulation and lung tissue that are characterized as ?hyperresponsive? in terms of cytotoxicity and cytokine responsiveness. We also examined earlier stages of disease using a mouse model employing a uterine specific Tsc2 deletion which develop hallmark features of LAM including leiomyoma formation and accumulation of pulmonary nodules consisting of smooth muscle cells carrying Tsc2 deletions. We show that the leiomyomas express high levels of ligands for the NKG2D activating receptor but also elaborate high levels of soluble ligands that likely downregulate NKG2D expression and impaired NK cell function. The preliminary data demonstrates a dynamic shaping of NK cell phenotype and function throughout the arc of pathogenesis. The goal of this proposal is to define the phenotype, function, and role of NK cells and NKG2D in LAM using both LAM patient samples and animal models. Towards this goal, we will 1) Define the functional significance of unique NK cell populations in LAM. 2) Define the mechanism of VEGFD-amplified NK cell activation. 3) Define the function of NK cells and Nkg2d in the initiation and progression of LAM. And 4) Define the efficacy and benefits of therapeutic inhibition of circulating soluble Nkg2d ligands. The successful completion of these studies is likely to have a high impact on our understanding of LAM pathogenesis. The role of cytotoxic lymphocytes such as NK cell are clear in terms of the multiple stages of disease such as the control of tumor growth and tissue remodeling. We will identify the role of these cells at multiple stages of disease, identify mechanisms of NK cell responsiveness and breakdown of immune surveillance, and examine the potential for therapeutic intervention targeting soluble NKG2D ligands. Several therapeutics aimed at modulating NK cell function are in development for chronic infections, autoimmune diseases, and cancer. Our hope is that these agents will provide new options for therapy, in addition to existing therapies for LAM, in the near future.